Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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A novel molecule from the arylalkylamine family of drugs, KHL-8430, has been identified as a potent and specific inhibitor of calmodulin activity. The effect of this drug on calmodulin-mediated enzymatic actions has been analyzed to exemplify how to model the mechanism of action of a functional calmodulin antagonist. The approach used includes both binding and enzyme kinetic studies. In both types of experiments, the effects of drugs on calmodulin-phosphofructokinase [ATP:D[fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11] and calmodulin-phosphodiesterase (3':5' cyclic nucleotide phosphodiesterase, EC 3.6.1.3) interactions have been investigated. We have found that KHL-8430, in contrast to trifluoperazine, a classical anticalmodulin drug, competes with neither phosphofructokinase nor phosphodiesterase for calmodulin binding, yet it liberates phosphofructokinase from calmodulin inhibition and phosphodiesterase from calmodulin stimulation. The anticalmodulin activity occurs at lower KHL-8430 than trifluoperazine concentrations. These findings might establish the functional importance of these differences in the specificity of these drugs. The synthesis of the data suggests that (i) whereas trifluoperazine antagonizes both phosphofructokinase and phosphodiesterase binding to calmodulin, KHL-8430 interacts with calmodulin complexed with enzymes; (ii) KHL-8430 binds to the calmodulin-phosphofructokinase complex with an affinity constant of 0.8 microM, whereas the binding constant of trifluoperazine is 2.5 microM (iii) within the ternary complex the dimeric form of the kinase preserves activity that is otherwise inactive; and (iv) the binding of trifluoperazine and KHL-8430 to calmodulin exhibits negative cooperativity. The approach used in this study makes it possible to screen for the calmodulin antagonist effect of other drugs as well.
Mol Pharmacol 1990 Dec
PMID:Dissimilar mechanisms of action of anticalmodulin drugs: quantitative analysis. 214 57

The dunce locus of Drosophila melanogaster codes for a low Km, cAMP phosphodiesterase. The correct function of this gene is required for normal learning and memory activity in flies, because dunce mutants fail in tests of behavioral conditioning. These observations have indicated that cAMP regulation is an important aspect of the biochemistry underlying learning and memory processes in insects. To determine whether the locus is functionally conserved in mammals, we have expressed dunce gene homologs from the rat in a yeast expression system. We find that the rat homologs encode low Km, cAMP phosphodiesterases similar to that coded for by the Drosophila dunce+ gene and, more importantly, that the mammalian enzymes are inhibited by rolipram and RO 20-1724, drugs with antidepressant properties. Surprisingly, the dunce-encoded phosphodiesterase was not inhibited by rolipram or RO 20-1724. These findings suggest that the phosphodiesterases, through their regulation of cAMP levels, influence learning and memory in insects and mood in mammals.
Mol Pharmacol 1990 Jan
PMID:Rat homologs of the Drosophila dunce gene code for cyclic AMP phosphodiesterases sensitive to rolipram and RO 20-1724. 215 12

Cyclic nucleotide phosphodiesterases (PDEs) from canine trachealis were characterized with respect to their kinetic properties, sensitivity to selective inhibitors, and subcellular distribution. Extracts from whole tissue homogenates were applied to DEAE-Sepharose anion exchange columns and eluted with a linear sodium acetate gradient. Three major peaks of PDE activity were resolved. The first (PDE I), which eluted at 0.2 M sodium acetate, was applied to a calmodulin (CaM)-Sepharose affinity column and resolved into CaM-insensitive and CaM-sensitive PDEs. The CaM-insensitive isozyme (PDE Ia) had apparent Km values of 135 microM (cAMP) and 4 microM (cGMP) and was potently inhibited by zaprinast (Ki = 0.1 microM). The CaM-sensitive isozyme (PDE Ic) had apparent Km values of 1 microM (cAMP) and 2 microM (cGMP) and was inhibited by zaprinast with an apparent Ki of 35 microM. The second peak of activity (PDE II) from the anion exchange column eluted at 0.3 M sodium acetate and had apparent Km values of 93 microM (cAMP) and 60 microM (cGMP). The enzyme displayed positive cooperativity with respect to the hydrolysis of cAMP (nH = 1.7). Low concentrations of cGMP (0.1-1 microM) reduced cooperativity (nH = 1.1) and increased the hydrolysis of 1 microM cAMP. The third peak of activity from the anion exchange column eluted at 0.6 M sodium acetate and displayed anomalous kinetics that suggested the presence of two isozymes. This was supported by the observation that enzyme activity was only partially inhibited by SK&F 94120 or Ro 20-1724 but was abolished by the combination of the two PDE inhibitors. Subsequent studies confirmed the existence of two isozymes. The first, PDE III, had apparent Km values of 0.3 microM (cAMP) and 8 microM (cGMP) and was inhibited by cGMP (IC50 = 0.1 microM), SK&F 94120 (Ki = 7.8 microM), and SK&F 94836 (Ki = 0.4 microM). The second, PDE IV, had apparent Km values of 4 microM (cAMP) and 40 microM (cGMP) and was inhibited by Ro 20-1724 (Ki = 5.2 microM) and rolipram (Ki = 0.5 microM) but not by cGMP. Assessment of the 100,000 x g soluble and particulate PDE activity revealed that all five isozymes were present in the soluble fraction, but only four isozymes (PDEs Ia, Ic, III, and IV) were present in the particulate fraction. These results indicate that five distinct PDE isozyme exist in canine trachealis and that these isozymes differ in their kinetic characteristics, sensitivity to activators and inhibitors, and subcellular distribution.
Mol Pharmacol 1990 Feb
PMID:Characterization and selective inhibition of cyclic nucleotide phosphodiesterase isozymes in canine tracheal smooth muscle. 215 70

Intracellular cyclic AMP (cAMP) regulates many critical differentiated functions of tracheal epithelial cells. An in vitro model system for reliable study of cAMP metabolism in these cells has been developed. Viable tracheal epithelial cells could be recovered from greater than 50% of necropsy specimens. Culture success rate was not significantly affected by age of subject, endotracheal intubation, or time between death and autopsy, although most specimens were obtained within 24 h of death. Human tracheal epithelial cells grown in primary culture displayed a typical histologic epithelial appearance, and the ultrastructure showed microvilli, junctional complexes, and tonofilaments. The cells uniformly stained with fluorescent antibody to cytokeratin, and expressed receptors for isoproterenol and vasoactive intestinal peptide. Human tracheal epithelial cells grown serum-free in an equal volume mix of Ham's F12 medium and Dulbecco's minimal essential medium containing growth supplements (Medium A) and cholera toxin (CT) had higher basal cAMP levels and greater increase in intracellular cAMP in response to phosphodiesterase inhibition than cells grown in Medium A without CT. Cells grown in Medium A without CT had similar morphology and grew at a comparable rate but attached to the culture substratum less readily than cells grown in Medium A with CT. Cells grown in Medium A without CT had less cAMP response to phosphodiesterase inhibition, less rapid accumulation of cAMP, and greater proportional response to receptor-mediated stimulation of cAMP production compared to cells grown with CT, though the final cAMP levels achieved were comparable.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1990 Jan
PMID:Adenosine 3:5' cyclic monophosphate synthesis by human tracheal epithelial cells. 215 12

Transforming Harvey (Ha) ras oncogene products accelerated the time course of Xenopus oocyte maturation induced by insulin, insulinlike growth factor 1, or progesterone. The transforming constructs, [Val-12]Ha p21 and [Val-12, Thr-59]Ha p21, displayed equal potency and efficacy in their abilities to accelerate the growth peptide-induced response. Normal Ha p21 was only 60% as powerful and one-fifth as potent as the mutants containing valine in the 12 position. In contrast, two nontransforming constructs, [Val-12, Ala-35, Leu-36, Thr-59]Ha p21 and [Val-12, Thr-59]Ha(term-174) p21, had no effect on the time course of hormone-induced maturation. Effects of the transforming ras proteins on hormone-induced maturation correlated with their abilities to stimulate in vivo phosphodiesterase activity measured after microinjection of 200 microM cyclic [3H] AMP. When p21 injection followed 90 min of insulin treatment, there was no increase in phosphodiesterase activity over that measured after hormone treatment or p21 injection alone, but additive effects of p21 and insulin on enzyme activity were observed during the first 90 min of insulin treatment. Even though normal Ha p21 and transforming [Val-12, Thr-59]Ha p21 stimulated oocyte phosphodiesterase to equal levels when coinjected with substrate at the initiation of the in vivo assay, the transforming protein elicited a more sustained stimulation of enzyme activity. These results suggest that stimulation of a cyclic AMP phosphodiesterase activity associated with insulin-induced maturation is involved in the growth-promoting actions of ras oncogene products in Xenopus oocytes.
Mol Cell Biol 1990 Apr
PMID:Transforming ras proteins accelerate hormone-induced maturation and stimulate cyclic AMP phosphodiesterase in Xenopus oocytes. 215 40

The cyclic nucleotide phosphodiesterase (phosphodiesterase) plays essential roles throughout the development of Dictyostelium discoideum. It is crucial to cellular aggregation and to postaggregation morphogenesis. The phosphodiesterase gene is transcribed into three mRNAs, containing the same coding sequence connected to different 5' untranslated sequences, that accumulate at different times during the life cycle. A 1.9-kilobase (kb) mRNA is specific for growth, a 2.4-kb mRNA is specific for aggregation, and a 2.2-kb mRNA is specific for late development and is only expressed in prestalk cells. Hybridization of RNA isolated from cells at various stages of development with different upstream regions of the gene indicated separate promoters for each of the three mRNAs. The existence of specific promoters was confirmed by fusing the three putative promoter regions to the chloramphenicol acetyltransferase reporter gene, and the analysis of transformants containing these constructs. The three promoters are scattered within a 4.1-kilobase pair (kbp) region upstream of the initiation codon. The late promoter is proximal to the coding sequence, the growth-specific promoter has an initiation site that is 1.9 kbp upstream of the ATG codon, and the aggregation-specific promoter has an initiation site 3 kbp upstream.
Mol Cell Biol 1990 May
PMID:The cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum contains three promoters specific for growth, aggregation, and late development. 215 67

We have isolated cDNA clones representing cyclic AMP (cAMP)-specific phosphodiesterases (PDEases) from a human monocyte cDNA library. One cDNA clone (hPDE-1) defines a large open reading frame of ca. 2.1 kilobases, predicting a 686-amino-acid, ca. 77-kilodalton protein which contains significant homology to both rat brain and Drosophila cAMP PDEases, especially within an internal conserved domain of ca. 270 residues. Amino acid sequence divergence exists at the NH2 terminus and also within a 40- to 100-residue domain near the COOH-terminal end. hPDE-1 hybridizes to a major 4.8-kilobase mRNA transcript from both human monocytes and placenta. The coding region of hPDE-1 was engineered for expression in COS-1 cells, resulting in the overproduction of cAMP PDEase activity. The hPDE-1 recombinant gene product was identified as a low-Km cAMP phosphodiesterase on the basis of several biochemical properties including selective inhibition by the antidepressant drug rolipram. Known inhibitors of other PDEases (cGMP-specific PDEase, cGMP-inhibited PDEase) had little or no effect on the hPDE-1 recombinant gene product. Human genomic Southern blot analysis suggests that this enzyme is likely to be encoded by a single gene. The presence of the enzyme in monocytes may be important for cell function in inflammation. Rolipram sensitivity, coupled with homology to the Drosophila cAMP PDEase, which is required for learning and memory in flies, suggests an additional function for this enzyme in neurobiochemistry.
Mol Cell Biol 1990 Jun
PMID:Cloning and expression of cDNA for a human low-Km, rolipram-sensitive cyclic AMP phosphodiesterase. 216 May 82

Although adenosine is known to activate K+ conduction in atrial tissue, there is still debate as to the involvement of cAMP-dependent mechanisms. In isolated adult guinea pig atrial myocytes, we demonstrate that the highly A1-selective adenosine receptor agonist 2-chloro-N6-cyclopentyladenosine reduced basal cAMP levels by 30-40% in the absence and presence of the nonxanthine phosphodiesterase inhibitor Ro 20-1724. Isoprenaline caused a concentration-dependent increase in cAMP levels, which was more pronounced in the presence of the phosphodiesterase inhibitor. Several adenosine derivatives suppressed the isoprenaline-induced cAMP increase by approximately 80%. The rank order of potency was 2-chloro-N6-cyclopentyladenosine (IC50, 93 nM) greater than (R)-N6-phenylisopropyladenosine (IC50, 309 nM) greater than 5'-N-ethylcarboxamidoadenosine (IC50, 813 nM) much greater than (S)-N6-phenylisopropyladenosine (IC50, 26,300 nM). A similar but complete suppression of the isoprenaline-induced cAMP increase was produced by the muscarinic receptor agonist carbachol (IC50, 398 nM), which like adenosine is known to activate atrial K+ channels. The A1-adenosine receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine antagonized the effect of 2-chloro-N6-cyclopentyladenosine concentration-dependently, with a KB value of 9.6 nM. In atrial myocytes isolated from guinea pigs pretreated with pertussis toxin, the inhibitory effects of adenosine analogs on basal and isoprenaline-stimulated cAMP accumulation were markedly attenuated. It is concluded that the adenosine receptor in guinea pig atrial myocytes, which is known to be linked to K+ channels, is also coupled to adenylate cyclase via a pertussis toxin-sensitive guanine nucleotide-binding protein and shows the characteristics of the A1-adenosine receptor subtype.
Mol Pharmacol 1990 Jun
PMID:Pharmacological characterization of the adenylate cyclase-coupled adenosine receptor in isolated guinea pig atrial myocytes. 216 17

Measurements of cyclic phosphodiesterase, or of beta-galactosidase in the case of cpdB'-'lacZ fusions, indicate that cpdB expression in both Escherichia coli and Salmonella typhimurium is modulated by carbon source availability, consistent with previous observations in Salmonella. Nucleotide sequence analysis and transcription mapping of both cpdB genes have revealed, in their 5' flanking regions, sequences with good similarity to consensus -10 and -35 regions and cyclic AMP-cyclic AMP receptor protein (cAMP-CRP) binding sites. Furthermore, they are strongly conserved in both organisms. Deletion analysis of an E. coli cpdB'-'lacZ fusion supports the identification of these elements, and a role for the cAMP-CRP binding site in modulating constitutive cyclic phosphodiesterase expression.
Mol Gen Genet 1990 Jun
PMID:Transcription and regulation of the cpdB gene in Escherichia coli K12 and Salmonella typhimurium LT2: evidence for modulation of constitutive promoters by cyclic AMP-CRP complex. 217 62

Caffeine potently inhibited forskolin-stimulated cyclic AMP accumulation in slices of rat cerebral cortex, with an IC50 of 21 +/- 3 microM. Because caffeine competitively blocks adenosine receptors, we examined whether the action of forskolin involved endogenous adenosine or whether caffeine was acting through some novel mechanism. Inhibition by caffeine was observed at all forskolin concentrations examined, although the degree of inhibition decreased at higher concentrations of forskolin. The effect of caffeine was not blocked by the presence of a phosphodiesterase inhibitor but was mimicked by several other methylxanthines. The most potent of these was 8-(p-sulfophenyl)-theophylline, which does not readily cross cell membranes, arguing for an extracellular site of action. Addition of either adenosine or the adenosine uptake blocker dipyridamole potentiated the forskolin response, suggesting that forskolin and adenosine act synergistically in increasing cyclic AMP accumulation. The nonxanthine adenosine receptor antagonist CGS 15943 potently blocked cyclic AMP responses to forskolin, adenosine, and combinations. 3-Isobutyl-1-methylxanthine potently blocked the response to adenosine but caused little or no inhibition of the response to forskolin. Adenosine deaminase (ADA) was added to eliminate contributions of endogenous adenosine. ADA inhibited the response to both adenosine and forskolin; however, 200 times as much enzyme was necessary to inhibit the forskolin response. Inhibition of added ADA with 2'deoxycoformycin dramatically increased the concentration of ADA required to inhibit the adenosine response, without altering the concentration required to inhibit the forskolin response. These results suggest that forskolin-stimulated cyclic AMP accumulation may be partially dependent on endogenous adenosine but that the inhibition observed with caffeine is not solely due to blockade of adenosine receptors.
Mol Pharmacol 1990 Nov
PMID:Is adenosine involved in inhibition of forskolin-stimulated cyclic AMP accumulation by caffeine in rat brain? 217 72


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